Macromolecules 2009, 42, 6723–6732
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DOI: 10.1021/ma9011818
Mimicking PAMAM Dendrimers with Amphoteric, Hybrid Triazine
Dendrimers: A Comparison of Dispersity and Stability
Sanjiv Lalwani,*,† Abdellatif Chouai, Lisa M. Perez, Vanessa Santiago, Sunil Shaunak,‡ and
Eric E. Simanek*,†
†
Department of Chemistry, Texas A&M University, College Station, Texas 77843, and ‡Faculty of Medicine,
Imperial College London, Hammersmith Hospital, Ducane Road, London, W12 ONN, U.K.
Received June 2, 2009; Revised Manuscript Received July 17, 2009
ABSTRACT: Two strategies are applied to mimic the amphoteric nature of the surfaces of half-generation
PAMAM dendrimers yet retain the very narrow dispersity inherent of triazine dendrimers. Both strategies
start with a monodisperse, single-chemical entity, generation two triazine dendrimer presenting 12 surface
amines that is available at the kilogram scale. The first method relies on reaction with methyl bromoacetate.
Complete conversion of the surface primary amines to tertiary amines occurs to provide 24 surface esters.
Extended reaction times lead to quarternization of the amines while other unidentified species are also
present. The resulting polyester can be quantitatively hydrolyzed using 4 M aqueous HCl to yield a dendrimer
with 12 tertiary amines and 24 carboxylic acids about a hydrophobic triazine core. The second method utilizes
Michael additions of methyl acrylate to yield 24 surface esters. This reaction proceeds more rapidly and more
cleanly than the former strategy. Hydrolysis of this material proceeds quantitatively using 4 M aqueous HCl
to yield the desired dendrimer. In both cases, MALDI-TOF mass spectrometry provides compelling
evidence of reaction progress. Electrophoretic analysis confirms the amphoteric nature of these materials
with the former targets having a pI value in the 1.8 < pI < 3.4 range, and the latter having a pI value in the
4.7 < pI < 5.9. These ranges bookend the pH range within which PAMAM dendrimers become zwitterionic,
3.4 < pI < 4.7. The strategy of using monodisperse amine-terminated dendrimer constructs as core offers
significant advantage over PAMAM homopolymers including dispersity, ease of characterization and batchto-batch reproducibility. These triazine dendrimers could ultimately be adopted into materials with
applications wherein the demands of purity have hitherto remained unsatisfied.
*To whom correspondence should be addressed. E-mail: simanek@
mail.chem.tamu.edu.
2,4,6-trichloro-1,3,5-triazine (cyanuric chloride), a low-cost and
readily available reagent. Using this chemistry, kilogram-scale
synthesis of a generation two triazine dendrimer12 has been
reported that used only a single chromatographic step and which
provided materials judged conservatively as being approximately
93% pure based on reversed-phase HPLC analysis of the BOCprotected intermediate. Higher purities can be achieved if intermediates are subjected to more rigorous purification than the
precipitation strategy utilized in the scale-up procedure. Recently, this material was used in the synthesis of anionic triazine
dendrimers derived from the succinylation of generation two and
generation three amine-terminated triazine dendrimers.13 Analysis of these materials using capillary electrophoresis (CE) in
comparison to the commercially available PAMAM analogues
showed a significant difference in molecular heterogeneity:
PAMAM dendrimers were highly disperse mixtures whereas
triazine dendrimers approached single-chemical-entity materials.
Full-generation (amine terminated) and half-generation
(carboxylate terminated) PAMAM dendrimers have shown
markedly different behavior in biological applications: in a report
by Malik et al., amine-terminated PAMAM dendrimers displayed generation-dependent hemolysis and changes in red cell
morphology whereas anionic dendrimers were neither hemolytic,
nor cytotoxic;14 El-Sayed et al. reported increased paracellular
permeability within a “size window” of generation 2.5 and 3.5
PAMAM carboxylate terminated dendrimers;15 an investigation
on the effect of PAMAM dendrimers on planar phosphatidyl
choline membranes by Shcharbin et al.16 showed that cationic
PAMAM generation 5 dendrimer disrupted the membranes
r 2009 American Chemical Society
Published on Web 08/12/2009
Introduction
The impact of polyamidoamine (PAMAM) dendrimers across
diverse fields including applications in therapeutics,1,2 diagnostics,3 sensing,4 material science,5 and catalysis6 since Tomalia’s
first report7 cannot be understated. Indeed, PAMAM or Starburst dendrimers are the most extensively studied family of these
materials.8 PAMAM dendrimers are commercially available with
amine terminal groups referred to as full-generation dendrimers,
or with carboxylate terminal groups referred to as half-generation
dendrimers. Full-generation dendrimers can have a cationic or a
neutral surface depending on the pH. Half-generation dendrimers have an amphoteric (and under certain pH conditions,
zwitterionic) surface. With these materials going into preclinical
trials9 precise knowledge of the structural complexity of these
dendrimeric nanodevices becomes increasingly important.10
Significant effort has been invested in characterizing these materials using a combination of corroborative methods.11 In general,
PAMAM materials exist as highly complex mixtures: the twostep iterative polyamidoamine chemistry utilized in PAMAM
dendrimer synthesis introduces the possibility of nonideal dendrimer growth through a variety of factors that affect the
monodispersity of the growing polymer.7 Retro-Michael reactions account for another source of structural heterogeneity.
As an alternative to PAMAM dendrimers, our efforts have
been toward providing single-entity dendrimers that can
be synthesized reproducibly and with high purity: we use
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whereas carboxylate-terminated PAMAM generation 4.5 dendrimer did not. From these demonstrated examples, and many
more, it is evident that the surface chemistry and topology of
dendrimers is a critical parameter in their applications-based
utility.
In this paper, we report the synthesis and characterization of
amphoteric triazine dendrimers obtained using two strategies.
The first scheme involves reacting methyl bromoacetate with
generation two amine-terminated triazine dendrimer, followed
by exhaustive hydrolysis of the ester groups. Two hydrolysis
conditions are tested: use of 4 M HCl and 5 M NaOH, both at
room temperature. The rates of ester hydrolysis and purity of
final products in the tested conditions are compared. Overall,
the first scheme yields a triazine dendrimer bearing 24 acetate
groups. Similarly, in the second scheme, generation two amineterminated triazine dendrimer is reacted with methyl acrylate
via Michael addition to result in a material with 24 surface
ester groups. Subsequent hydrolysis of this material was
affected in 4 M aqueous HCl and 5 M NaOH. Overall, the
second scheme resulted in a triazine dendrimer displaying
24 propionate groups. From our work on the succinylation
of triazine dendrimers13 it was observed that progress of the
succinylation reaction can be meticulously followed using
MALDI-TOF MS and CE: mass spectrometry identifies the
species present in the mixture at the end of the reaction and CE
provides quantitative information on purity. As such, the
synthesis of the ester-bearing intermediates and the carboxylate-bearing final products was closely monitored using
MALDI-TOF MS. Purity and molecular heterogeneity assessments were obtained using CE and were compared with
commercially available PAMAM analogues. PAMAM dendrimers with the diaminobutane (DAB) core were selected for
comparison instead of ethylenediamine (EDA) core analogues
because of their inherent interest to the group, their broader
spectrum of use, and lower dispersity as indicated by slab gel
electrophoresis in the available product literature.
Experimental Section
Materials. All reagents and solvents needed in the synthesis
and the analysis of triazine dendrimers were purchased from
Aldrich Chemical Co. (Milwaukee, WI) and were used as
received. Uncoated fused silica capillaries were purchased from
Polymicro Technologies, LLC, (Phoenix, AZ). Commercial
half-generation, diaminobutane-core PAMAM carboxylate
dendrimers were also purchased from Aldrich Chemical Co.
The second generation triazine dendrimer used in this work was
synthesized in our laboratory as previously reported.12
Synthesis of 1e. A solution of the generation two amineterminated triazine dendrimer was prepared by dissolving 50.0
mg (0.0169 mmol) of the solids into 2.0 mL of the appropriate
solvent (methanol or tetrahydrofuran). To this was added
methyl bromoacetate (37.2 mg, 22.4 μL, 0.244 mmol) corresponding to 1.2 molar equiv per NH2. The reaction mixture was
allowed to stir at room temperature for 2 h. Formation of a
white precipitate was observed (presumably the hydrobromide
salt of the partially substituted dendrimer). Then, 68.7 μL of a
separately prepared methanolic solution of potassium hydroxide (prepared by dissolving 206 mg of solid potassium hydroxide in 1.0 L of methanol under rigorous stirring) was added to
the dendrimer-containing reaction mixture. The mixture was
stirred at room temperature and sample was taken for analysis
by MALDI-TOF MS after 14 h of reaction time. Further
additions of 1.2 molar equiv of methyl bromoacetate per NH2
were required, each one accompanied by the addition of 68.7 μL
of the methanolic potassium hydroxide solution. Progress of the
reaction was monitored using MALDI-TOF MS. A total of 8.4
molar equiv of methyl bromoacetate per NH2 had been
added when signals corresponding to 25- and 26-ester-bearing
Lalwani et al.
dendrimers were observed along with the desired 24-ester-bearing species, as well as signals corresponding to incompletely
substituted dendrimers.
13
H NMR (CDCl3): δ 5.35-4.70 (br m), 4.70-3.30 (br m),
3.30-2.89 (br m), 2.67-1.00 (br m). 13C NMR (CDCl3): δ
175.9-163.6, 125.6, 61.7-59.7, 55.4-54.2, 52.7-51.2, 47.342.6, 41.5-38.5, 30.3, 29.7, 28.3-24.1.
Synthesis of 1a. An aliquot of the reaction mixture corresponding to 10.0 mg of dendrimer 1e was taken, and the solvent
was removed under reduced pressure. The glassy material that
remained was dissolved in 400 μL of 4 M HCl and allowed to
react at room temperature. Progress of the hydrolysis reaction
was monitored using MALDI-TOF MS. A 200 μL aliquot of
the solution was then transferred into a preconditioned Centricon centrifugal filter device (Amicon Bioseparations) having
a regenerated cellulose membrane with a 3,000 molecular weight
cutoff. The retained solution was collected and used as is for
further analysis.
1
H NMR (D2O): δ 4.00-2.70 (br m), 2.60-2.15 (br s), 2.040.41 (br m). 13C NMR (D2O): δ 179.8-178.3, 167.1-163.7,
59.5-57.9, 54.4-51.3, 45.5-41.4, 26.4-23.6.
Synthesis of 2e. A solution of the generation two amineterminated triazine dendrimer was prepared by dissolving 300
mg (0.101 mmol) of the solids into 5.0 mL of methanol under
constant stirring. To this solution, 252 mg (263 μL, 2.92 mmol)
of methyl acrylate was added slowly, corresponding to 2 molar
equiv per NH2 and a corresponding 20% mol/mol excess per
addition. The reaction mixture was allowed to stir at room
temperature. Samples were taken over time and analyzed by
MALDI-TOF MS. Reaction was deemed complete after 3 days
of reaction time.
1
H NMR (CDCl3): δ 4.00-3.00 (br m), 2.90-2.00 (br m),
1.97-1.13 (br m). 13C NMR (CDCl3): δ 173.4-172.6, 166.5164.3, 125.5, 51.9-50.9, 49.4-48.5, 45.8-44.7, 44.6-43.5,
43.4,-42.6, 39.2-36.4, , 32.7-31.8, 30.2, 28.6-27.1, 26.4-24.6.
Synthesis of 2a. An aliquot of the reaction mixture corresponding to 10.0 mg of dendrimer 2e was taken, and the solvent
was removed under reduced pressure. The glassy material that
remained was dissolved in 400 μL of 4 M HCl and allowed to
react at room temperature. Progress of the hydrolysis reaction
was monitored using MALDI-TOF MS which showed complete hydrolysis of the esters within 2 days. A 200 μL aliquot of
the solution was then transferred into a preconditioned Centricon centrifugal filter device (Amicon Bioseparations) having
a regenerated cellulose membrane with a 3000 molecular weight
cutoff. The retained solution was collected and used for further
analysis as is.
1
H NMR (D2O): δ 4.19-2.77 (br m), 2.76-2.22 (br s), 2.200.41 (br m). 13C NMR (D2O): δ 177.9-175.5, 167.0-163.5,
51.6-47.9, 46.0-41.1, 30.3-28.0, 26.2-23.6, 23.3,-20.6.
Capillary Electrophoresis. A P/ACE MDQ capillary electrophoresis system with a photodiode array detector (BeckmanCoulter, Fullerton, CA) was used to obtain all of the CE
separations. Uncoated fused silica capillaries were treated according to the coating procedure described by Kaneta et al.17
using poly(vinylpyrrolidone) as a semipermanent coating.
Capillaries used had an internal diameter of 50 μm and typical
lengths as follows: total length of the capillary, Lt, 30.6 cm;
length of the capillary from injection point to the detection
window, Ld, 20.7 cm. Lengths Lt and Ld varied from cartridgeto-cartridge by (0.5 cm.
The different background electrolytes (BGEs) used were
made by first preparing a solution of the buffering species of
the desired concentration, followed by titration to the desired
pH using the titrant in the pure form (as solid or liquid). Further
details on the selection of BGE components and preparation are
provided as part of the Supporting Information. Solutions of
0.2-0.5% dimethyl sulfoxide (DMSO) in water were used as the
neutral marker. All purity and dispersity analysis were performed using conventional CE protocols (i.e., capillary rinse;
Macromolecules, Vol. 42, No. 17, 2009
Article
pressure injection of sample; optional pressure injection of
neutral marker; electrophoresis) whereas determination of pH
ranges which bracket the respective isoelectric points of the
dendrimers were determined by pressure-mediated capillary
electrophoretic (PreMCE) separations.18
MS Characterization. MALDI-TOF mass spectra were performed on a ABI Voyager-DE STR mass spectrometer operating in reflected mode using 2,4,6-trihydroxyacetophenone
(THAP) as matrix. When needed, MS data was reprocessed
using Origin 8 and Microsoft Excel software packages.
NMR Spectroscopy. 1H and 13C NMR spectroscopy was
performed using either Mercury 300 or an Inova 300 instrument.
Since broad and overlapping signals are observed, chemical
shifts are reported as ranges.
Computation. Computational results were obtained using the
software package Materials Studio (MS) 4.4 (Accelrys, Inc.).
The dendrimers were built in the fully extended conformation
using the dendrimer builder tools in MS. The conformational
space of each dendrimer was sampled via 300 Simulated Annealing (SA) cycles over a period of 840 ps using the Forcite Plus
program and the polymer consistent force-field (pcff) as implemented in MS 4.4. The SA runs utilized constant volume
and temperature (NVT) molecular dynamics (MD) over a
temperature range of 300-1000 K using the Nose thermostat,
ΔT = 50 K, untruncated atom-based electrostatic and van der
Walls interactions, and a time step of 1 fs. The dendrimer was
minimized after each annealing cycle, resulting in 300 minimized
structures per dendrimer. Electrostatic potential maps were
generated at the AM1 level of theory using the vamp program
as implemented in MS for the lowest energy conformation of
each dendrimer obtained from the SA simulations.
Results and Discussion
Nomenclature. Scheme 1 shows the synthesis of the target
dendrimers from a previously reported, generation two
dendrimer with 12 amine groups. Reaction with methyl
bromoacetate yields 1e, a generation 2.5 (adopting the
PAMAM nomenclature) hybrid dendrimer wherein the “e”
reflects the ester periphery.12 Hydrolysis yields the polyacid,
1a. Similarly, reaction with methyl acrylate yields 2e that is
subsequently hydrolyzed to polyacid 2a.
Synthesis of 1e and 1a. Reaction of the generation two
amine-terminated triazine dendrimer with methyl bromoacetate was attempted in two solvents, methanol and tetrahydrofuran. Aliquots were removed to monitor the progress
of the reaction by MALDI-TOF MS. Figure 1 shows the
traces obtained for the reaction in methanol. The starting
material shows a single line at m/z 2954. Panels A and B
capture the reaction at 3 h and 1 day. The characteristic
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separation of 72 mass units corresponding to the primary
ladder of peaks represents additional “CH2COOCH3”
groups. The numbers above the peaks correspond to the
number of additions. The suggestion of greater complexity in
panel B and clear presence in panel C results from (i) the
detection of both proton and potassium adducts and (ii)
undesired ester hydrolysis. Potassium adducts are prominent
due to the use of KOH as a base in the reaction. Finally, panel
D provides clear warning against the overinterpretation of
mass data. Here, as a function of ionization conditions, it is
possible to obtain spectra that show intermediates wherein
only an even number of substitutions have occurred including prominent lines at 18, 20, 22, and the desired 24 substitutions. Optimizing ionization conditions allows for the
expected spectra. Curiously, we are unable to suppress the
even products in favor of the odd as a function of ionization
conditions or matrix. The Supporting Information details
some of these matrix effects, and as a result, we decided to use
2,4,6-trihydroxyacetophenone (THAP) throughout this
study. The spectrum shown in panel E reveals a limitation
to this chemistry. At increasing reaction times, an additional
species that we attribute to a cyclization product (lactam)
becomes increasingly abundant.
As eluded to, closer inspection of the data reveals an even
richer description of the chemistries (Figure 2). Again,
numbers above the peaks correspond to the number of
methyl acetate units on the molecule. Four signals are
observed for each substitution: peaks corresponding to the
proton adduct (labeled as a) and potassium adduct (labeled
as c) in addition to peaks labeled as b and d at m/z values of
14-less than signals labeled as a and c respectively, corresponding to the loss of a methyl group (hydrolysis). These
lines result from the use of a methanolic solution of potassium hydroxide to scavenge the hydrobromic acid produced
in the reaction. Another potential side reaction, the substitution of the bromide functionality in the unreacted methyl
bromoacetate with a hydroxyl group also occurs. Although
this would not affect the number of signals observed in the
mass spectrum, the occurrence of such side reaction is
evident from the amount of excess reagent required to drive
the reaction to completion.
Further, the nature of the chemistry employed does not
provide sufficient control over the formation of tertiary
amine versus quaternary ammonium bearing products as
evident from mass spectra for the reaction in tetrahydrofuran obtained after 5 days of reaction time at room temperature (Figure 3, bottom trace). While complete substitution
has not yet been achieved, as revealed by lines corresponding
Scheme 1. Synthesis of the Hybrid Dendrimers
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Lalwani et al.
Figure 2. MALDI-TOF MS trace for an aliquot of the reaction
mixture for the synthesis of 1e. The number above the set of peaks
represents the number of methyl acetate substitutions on the generation
two triazine dendrimer. The legend for the corresponding peaks a-d is
indicated as an inset in the spectrum.
Figure 1. MALDI-TOF MS traces for aliquots taken from the reaction
mixture for the synthesis of 1e using methanol as the solvent. Number
above the peaks represents the number of methyl acetate substitutions on
the generation two triazine dendrimer. The corresponding reaction times
are as follows: (A) 3 h; (B) 1 day; (C) 5 days; (D) 6 days; (E) 11 days.
to 22 and 23 methyl acetate substitutions in addition to the
desired product, we also observe signals corresponding to
products with 1 and 2 quaternary ammonium groups. Just as
Figure 3. MALDI-TOF MS traces for aliquots taken from the reaction mixture for the synthesis of 1e using tetrahydrofuran as the solvent.
Spectrum obtained after 1 day of reaction time (top trace) shows 15-24
substitutions on the generation two triazine dendrimer (marked with “*”)
and signals decreasing by steps of m/z = 14 corresponding to increasing
ester hydrolysis. Spectrum obtained after 4 days (middle trace) shows
prevalent signals for species with even number of substitutions whereas
signals for species with an odd number of substitutions appear to be
disfavored. Spectrum obtained after 5 days (bottom trace) shows
products ranging from 22 to 26 substitutions (abbreviated as 22S to
26S), derivatives due to ester hydrolysis and potassium adducts thereof.
in the previous case, complicated spectra due to undesired
ester hydrolysis (Figure 3, top trace) and varying ionization tendencies are also observed in these samples, as
evident from the change in the relative intensities of signals
corresponding to species with odd- and even-numbered
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Figure 4. CE traces for 1e (bottom trace) and 2e (top trace) obtained
using a pH 3.4 100 mM formate/lithium buffer.
additions of methyl bromoacetate (top and middle traces in
Figure 3).
A sample from the tetrahydrofuran-containing reaction
mixture was analyzed by capillary electrophoresis, CE, using
a pH 3.4, 100 mM formate/lithium background electrolyte
(BGE). Under these conditions, 1e and other dendrimeric
products of the reaction are expected to migrate as cations.
Electrophoresis was therefore performed using a positive-tonegative polarity setup, where the positively charged electrode is placed near the point of injection and the negatively
charged electrode is placed near the outlet end of the
capillary. The bottom trace in Figure 4 shows the electropherogram. The observed peak width for 1e indicates a more
diverse composition than 2e. This is in agreement with results
of MALDI-TOF MS analysis, which indicates the presence
of both incompletely substituted dendrimer products and
those containing quaternary ammonium groups. Further,
CE provides hints toward the structural diversity of this
mixture. Due to the use of a pH 3.4 BGE for analysis using
CE, resolution between species having a different number of
ester groups is not expected, since the cationic character of
the molecule does not change (number of charge-bearing
amines remains constant). This is also true when quaternary
ammonium groups are formed, as even the secondary amines
are expected to be fully protonated at a pH of 3.4. However,
the cationic character of the dendrimer can decrease by two
paths: (i) ester hydrolysis and (ii) amide bond formation with
either the free methyl bromoacetate or intramolecular cyclization. In both cases, cations having a migration velocity
slightly slower than the desired product 1e will be generated.
Incomplete resolution of these dendrimers will result in a
greater peak width. We believe both of the mentioned
processes contribute to the observed dispersity.
Products from the interrupted synthesis of 1e were subjected to hydrolysis in 4 M HCl solution at room temperature. Aliquots were taken after 1, 4, 10, and 22 days of
reaction and analyzed by MALDI-TOF MS (Figure 5).
After 10 days, significant incomplete hydrolysis of the
esters was observed (data not shown) whereas by the 22nd
day only the completely hydrolyzed and 1-ester-group-bearing species are observed. The pattern showing dendrimer
species with three different degrees of substitution as seen in
the initial sample was preserved throughout the course of the
hydrolysis reaction. Intentional hydrolysis in 5 M NaOH
Figure 5. MALDI-TOF MS traces for aliquots of the reaction mixture
wherein 1a is produced from the hydrolysis of the esters of 1e recorded
after reaction times of 1 day (top trace), 4 days (middle trace) and 22
days (bottom trace). The spectra reveal different degrees of substitution:
sets of signals corresponding to triazine dendrimers with 20, 22, and 24
substitutions are observed. Within each set, peaks separated by an m/z
of 14 indicate an increasing number of ester groups still present.
proceeded much more rapidly: complete hydrolysis was
observed in two days at room temperature as determined
using MALDI-TOF MS (data shown as part of Figure 9).
Overall, it appears unlikely that single-entity triazine dendrimers can be obtained using this chemistry. Nonetheless, as
shown in the MS and CE analysis of the final products, the
synthesized triazine dendrimers are still of significantly higher purity in comparison with the commercially available
PAMAM analogues.
Synthesis of 2e and 2a. While reaction with methyl bromoacetate produces materials that resemble the structure of
PAMAM dendrimers, using methyl acrylate exactly mirrors
PAMAM chemistry. Indeed, the strategy proves remarkably
clean and straightforward. Samples of the reaction between
the triazine dendrimer and methyl acrylate were analyzed
over a 3-day period using MALDI-TOF MS. Figure 6
shows the traces that were obtained. As a consequence of
starting with a well-characterized amine-terminated dendrimer, the addition of each methyl acrylate unit onto the
dendrimer can be observed (panel A recorded after 1 day
of reaction time). Mass spectrum obtained after a 3 days at
room temperature is very simple and easy to interpret: one
major signal corresponding to a dendrimer 2e with 24
propionate esters and two minor signals corresponding to
species with 22 and 23 substitutions (panel B).
This choice of chemistry offers several advantages. First,
due to the nature of the Michael reaction, addition of base to
the reaction is not required, and as such, hydrolysis of the
ester groups is not observed. Also, a large excess of the active
reagent (methyl acrylate) is not needed: the reaction proceeds
close to completion with just 20% mol/mol excess reagent
(per addition) within the 3-day reaction time at room temperature.
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Lalwani et al.
Figure 6. MALDI-TOF MS traces for aliquots of the reaction mixture for the synthesis of 2e (panels A and B) and for the synthesis of 2a (panels C and
D). Peaks in panel A are separated by an m/z of 86 indicating an increasing number of methyl acrylate additions; panel B shows dendrimer with 23 and
24 additions. Inset in panel C shows peaks separated by an m/z of 14 indicating incomplete hydrolysis of the ester groups. Lastly, panel D shows two
peaks separated by an m/z of 72, indicating products with 23 and 24 carboxylic acids.
Figure 7. MALDI-TOF MS spectra of generation 1.5 (left panel) and generation 2.5 (right panel) PAMAM dendrimers bearing 16 and 32 carboxylic
acid groups, respectively. The number of carboxylic acid groups is based on an ideal structure and signals with lower mass units observed may be due to
dendrimers with structural defects.
A sample of the reaction mixture from the synthesis of 2e
was analyzed using CE. Identical conditions to those described earlier were used: a pH 3.4, 100 mM formate/ lithium
solution as BGE and a positive-to-negative polarity setup.
Under the chosen conditions, the desired product 2e and
other products of the reaction are expected to migrate as
cations. The bottom panel in Figure 4 shows the obtained
electropherogram. The single and narrow peak observed, in
comparison to the first derivative, 1e (top panel), must be
treated with tempered enthusiasm as there are limitations to
this analysis: the method is unable to resolve species having a
different number of ester groups unless accompanied by a
different number of amines (as is the case with amide
formation). Indeed, MALDI-TOF MS indicates the presence of dendrimers substituted 22 and 23 times. However,
these derivatives are not resolved by our CE method.
Hydrolysis was affected by a strong acid instead of a
strong base to limit retro-Michael reactions. Hydrolysis in
4 M HCl solution at room temperature was followed using
MALDI-TOF MS and was found to proceed cleanly: the
three-peak-pattern for dendrimers with different number of
added methyl acrylates observed prior to hydrolysis was seen
in both an intermediate sample (Figure 6, panel C) and the
final sample upon complete hydrolysis (Figure 6, panel D).
The spectrum for the final sample showed a major signal
corresponding to the dendrimer 2a with 24 propanoic groups
as well as the side products, dendrimers with 22 and 23 acids.
These materials prove to be much cleaner than the corresponding PAMAM dendrimers available to us commercially
(Figure 7) as observed using both MALDI-MS and capillary
electrophoresis (Figure 8).
Comparison of Dispersity. The dispersity of the synthesized hybrid triazine dendrimers and the commercially available PAMAM analogues was compared by two means:
MALDI-TOF MS and CE. Shown in Figure 7 are the MS
traces obtained for generation 1.5 and generation 2.5 diaminobutane core-PAMAM dendrimers. On the basis of the
ideal structure of the dendrimer, signals corresponding to
m/z values of 2610 and 5588 are expected for the two
materials. However, the obtained MS traces show a significantly greater number of signals in the 2200 to 2800 and the
3400 to 6500 m/z ranges for the two dendrimer samples,
respectively, indicating a complex mixture of dendrimer
molecules having nonideal structures. The notion that PAMAM analogues of the hybrid triazine dendrimers reported
in this work are highly complex mixtures is further corroborated by analysis using CE. Figure 8 shows the electropherograms obtained for the four dendrimers analyzed in this
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Macromolecules, Vol. 42, No. 17, 2009
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Figure 8. Electropherograms for the dendrimers included in this study obtained using a pH 6.9, 50 mM phosphate/lithium BGE in negative-to-positive
polarity: 1a (top trace), 2a (second trace), PAMAM G1.5 (third trace), and PAMAM G2.5 (bottom trace).
Figure 9. MALDI-TOF MS traces for 1a (left) and 2a (right) obtained to determine their stability under relatively harsh conditions: top traces were
obtained from samples immediately after hydrolysis of the corresponding ester precursors; middle traces were obtained after storage in 4 M HCl and
bottom traces were obtained after storage in 5 M NaOH (signals in the bottom traces separated by an m/z of 22 result from species with multiple sodium
counterions).
work. Analysis was performed using a pH 6.9, 50 mM
phosphate/lithium buffer using negative-to-positive polarity
(negatively charged electrode is placed near the injection end
of the capillary and positively charged electrode is placed
near the outlet end). The choice of BGE was guided by our
previous work on the development of a CE method for
analysis of anionic dendrimers.13 Unlike the CE analysis of
1e and 2e where a pH 3.4 BGE was used, here we expect to
be able to resolve species based on differences in both the
cationic and anionic characters is of the dendrimer.
The drastic difference in dispersity between triazine and
PAMAM dendrimers is obvious: traces for 1a and 2a (top
and second from the top respectively) show a major component and some minor components whereas traces obtained
for generation 1.5 and generation 2.5 PAMAM dendrimers
(second from the bottom and bottom trace respectively)
show a highly complex mixture of species where it becomes
difficult to determine which of the observed signals represents the dendrimer having the ideal structure.
Furthermore, we were interested in quantifying the
purity of the ideal dendrimer 2a as this proved to be present
as the least disperse mixture. Although the pH 6.9 phosphate/lithium buffer provided sufficient resolution, the fear
of interference from the “system peak” (aka eigenpeak)
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Macromolecules, Vol. 42, No. 17, 2009
generated close to the dendrimer signals prevented reliable
integration. On the basis of simulation experiments using the
Peakmaster software package19 an alternative BGE was
determined: a 100 mM, pH 6.5 BisTris/acetate BGE did
not generate system peaks with mobility values close to that
of the analyte. Using this BGE, the resolution remained
unchanged and the major signal in the trace for dendrimer 2a
was found to integrate to a corrected area of ∼80% relative
to the other observed signals (data not shown). Integration
of signals was not relevant for the other derivatives due to the
dispersity and/or low resolution, and thus further analysis on
these samples was not performed.
Comparison of Stability. Peterson et al. showed that
PAMAM half-generation dendrimers are highly susceptible
to degradation in the þ50 °C to -15 °C temperature range.20
Our objective was to determine the structural integrity of the
synthesized hybrid dendrimers in two extremely harsh conditions: 4 M aqueous HCl solution and 5 M NaOH solution.
Shown in Figure 9 are the MS traces obtained for the two
samples. Each panel has been labeled to indicate the corresponding dendrimer and consists of three traces representing: initial sample (top), sample stored in 4 M HCl (middle
trace) and sample stored in 5 M NaOH (bottom trace). It is
evident from the preserved three-species-pattern, based on
the number of substitutions on the triazine core, for the
hybrid dendrimer 1a that noticeable degradation has not
occurred in either of the two conditions. In the case of the
hybrid triazine dendrimer 2a, species corresponding to less
than 22 substitutions are not observed, and only a slight
change in the relative intensities of signals is seen, regardless
of whether the sample was stored for 21 days in 4 M HCl
(middle trace) or for 3 days in 5 M NaOH (bottom trace),
although the latter conditions lead to multiple sodiated
species. The slight change in relative intensities may be due
to the conditions favoring degradation via retro-Michael
reactions or may simply be an artifact attributed to possible
changes in the efficiency of ionization in MS.
Comparison of pI Values. To compare the inselectric point
(pI) values of 1a and 2a with their PAMAM analogues, the
method of Glukhovskiy and Vigh21 was adopted, where
pressure-mediated CE (PreMCE) was used to determine
the mobility of the dendrimers in BGEs having different
pH values. Both the effective mobility of the analyte and the
Lalwani et al.
mobility due to the electroosmotic flow can be calculated
from the traces obtained using this method. However, since
the objective was to compare the charge state of these
dendrimers under different pH conditions, a simple determination of whether the analyte band was moving as an anion
or as a cation suffices. The charge state on the dendrimer can
be inferred from the direction in which it migrates with
respect to a neutral marker as a result of the applied
potential: under our chosen conditions, if the distance between the bands increases, it can be inferred that the analyte
migrated as a cation. If the opposite is true, the analyte
migrated as an anion. Additional details of the technique are
provided in the Supporting Information.
Our previous work13 established that half-generation PAMAM dendrimers migrate as anions at pH 6.9. Accordingly,
we expect pI values for these dendrimers and their triazine
analogues to be less than 6.9. The mobility of these dendrimers under varying conditions using BGEs with pH values of
1.8, 3.4, 4.7, and 5.9 were determined (details as to the
formulation of the BGEs are given in the Supporting Information). Figure 10 shows the obtained PreMCE traces
(only relevant portions shown), separated into panels corresponding to the BGE used. Each trace consists of two signals:
the first trace in each panel shows two signals corresponding
to two distinct neutral marker bands within the capillary
whereas the first signal in succeeding traces corresponds to
the dendrimer being analyzed and the second signal corresponds to the neutral marker acting as a mobility reference.
A common artifact in all of the traces is a “system peak” (aka
eigenpeak) which appears at the same location as a neutral
marker. In the presence of a neutral marker, this artifact is
not observed, however, in the absence of a neutral marker (as
in the case of the dendrimer samples) this system peak
appears as a dip overlapping with the signal corresponding
to the dendrimer making it appear as if the analyte band was
split with some of the components migrating as cations and
others as anions.
Traces that were obtained using a pH 5.9 BGE are shown
in the first panel from the left. All of the dendrimers migrate
as anions. When a pH 4.7 BGE is used (second panel from
the left), triazine dendrimer 2a migrates as a cation, whereas
all other dendrimers continue to migrate as anions, indicating the pI for 2a is in the 4.7 < pI < 5.9 range. Use of a pH
Figure 10. Traces obtained using pressure-mediated capillary electrophoresis for PAMAM and triazine dendrimers at different pH values: pH 5.9 (first
panel), pH 4.7 (second panel), pH 3.4 (third panel) and pH 1.8 (fourth panel). Neutral marker signals are labeled as “N”; dendrimer signals are labeled
with an asterisk. Dashed line from the centroid of the first neutral marker in the first trace of each panel serves as a guide: analytes that migrate to the left
of the line migrate as cations, those migrating to the right migrate as anions. The dip observed in-line with the first neutral marker results from a
comigrating system peak.
Macromolecules, Vol. 42, No. 17, 2009
Article
6731
Table 1. Summary of Physical and Computational Dataa
a
The cores of the dendrimers are shown in red and the terminal carboxylic acid groups shown in green. Stick representations show the lowest energy
structure obtained from the SA calculations. AM1 electrostatic potential maps range from -0.2 kcal/mol (red) to 0.2 kcal/mol (blue) mapped on the
0.02 Å-3 electron density surface. The number of amine and carboxylate groups are based on idealized structures for the targets. Triazinyl amines are
counted to include the exocyclic nitrogen and aromatic ring nitrogen as a single unit, a strategy that undercounts the nitrogen atoms by 2, but may
overcount proton-accepting sites by 3 if the triazine unit is only protonated once.
3.4 BGE (third panel) places the pI value for the PAMAM
dendrimers in the 3.4 < pI < 4.7 since PAMAM dendrimers
migrate as cations in this BGE. Triazine dendrimer 1a
migrates as an anion in BGEs with pH values above 3.4,
and migrates as a cation in a pH 1.8 BGE (shown in the last
panel) and so its pI value must fall within the 1.8 < pI < 3.4
range. Having such low pI values is an important property
for these dendrimers as this results in a net-anionic charge on
the respective dendrimers under neutral conditions, bestowing upon them high aqueous solubility, such as that observed
for many proteins with pI values greater than or less than 7
(e.g., human albumin, a common protein in serum has a pI of
4.722).
The difference in pI values between the different dendrimers can be explained based on the structure of the molecules. On the basis of the difference between the pKa values
for the carboxylic acids of glycine (2.36)23 and β-alanine
(3.53)23 triazine dendrimer 1a is expected to have the lowest
pI value among all of the dendrimers tested. The two
PAMAM dendrimers are expected to have similar pI values:
in both cases the number of amines is equal to two less than
the number of carboxylic acids and the pKa values for both
the carboxylic acids and amines are not expected to be
significantly different. The triazine dendrimer 2a however
is expected to have a higher pI value than the PAMAM
dendrimers due to the additional contribution to the basic
(and cationic) character of the dendrimer coming from the
protonation of the triazine ring nitrogens (pKa for the ring
nitrogens of melamine, a model compound, is 5.124).
Computation. To complete this comparison of PAMAM
and hybrid triazine dendrimers, computation analyses were
performed. Table 1 summarizes the results of these studies as
well as a summary of other physical characteristics. All four
molecules are sufficiently small that the red core (triazine or
diaminobutane) groups have access to the surface. The acid
groups on the periphery are shown in green and hydrogenbonding interactions between these groups as well as with the
backbone are observed. Both the gas-phase diameters of
these species and the amount of free volume within the
structure are visually similar. The calculated radii of gyration
(reported as diameter) corroborate this expectation,
although G1.5 PAMAM is a flattened structure that leads
to a smaller number. This porosity is perhaps the only clue to
the nonproteinaceous (that is, well-organized and tightly
packed) nature of these macromolecules. Triazine groups
of the hybrid dendrimers are readily evident in yellow/orange
hues on the electrostatic potential maps. The high degree of
similarity in these structures leads us to hypothesize that
these hybrid dendrimers could serve as well-defined surrogates for low generation PAMAM materials. This similarity,
however, must be taken with some caution, as it derives from
gas-phase calculations. While valuable, these calculations
invariably lead to densely packed structures that may
not adequately communicate differences in solution. Such
differences might be expected due to the different hydrophobicity of the interiors of the hybrid and native PAMAM
structures. In preliminary studies, we have established that
the relative accessibility of the peripheral groups is similar as
judged by synthetic transformation. In due course, studies
where these hybrid structures are elaborated with additional
layers of PAMAM chemistry will be communicated.
Conclusion
Two high-purity triazine dendrimers that mimic the amphoteric surface of half-generation PAMAM dendrimers have been
synthesized. Capillary electrophoresis and MALDI-TOF MS
have been successfully employed in monitoring the progress of
the reactions and determination of purity and dispersity. Capillary electrophoresis was also used to determine the pH range
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Macromolecules, Vol. 42, No. 17, 2009
within which fall the pI values of the respective dendrimers
different between the two dendrimers. The triazine dendrimers,
2a appears to be the preferred choice: the design exactly mirrors
that of PAMAM dendrimers, synthesis proceeds with relative
ease and final products are of significantly higher purity, approaching single entity systems. Although the synthesis of the
dendrimer 1a proceeds with more difficulty and results in a
mixture of products, both capillary electrophoresis and MS
confirm the dispersity to be much lower than that of commercially available PAMAM mixtures. Further, having pI values less
than 7 provides sufficient aqueous solubility for all of the
dendrimers under neutral and/or biological conditions. Overall,
we believe the new hybrid triazine-PAMAM dendrimers could
serve as alternatives to the widely studied and useful, but highly
disperse commercially available PAMAM dendrimers.
Acknowledgment. This work was supported by the NIH
(U01 5U01AI075351-02; R01 GM64560-07). We would like to
thank the Laboratory for Molecular Simulation at Texas A&M
University for providing software and computer resources.)
Lalwani et al.
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Supporting Information Available: Text discussing the
selection of the BGE composition and preparation of BGE
solutions, optimization of the MS analysis, and a detailed
description of the 3-band PreMCE method and figures showing
MS spectra, a plot of the band positions, and complete PreMCE
traces recorded. This material is available free of charge via the
Internet at http://pubs.acs.org.
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